Screening And Identification Analysis Of Serum Protein Biomarkers In Nephroblastoma

BackgroundNephroblastoma or Wilms tumor (WT) is one of the most common malignant substantive tumor in pediatric surgery .The phases and sub-type is an important factor affecting the prognosis. Early diagnosis of nephroblastoma is of great significance to its treatment and prognosis. Nowadays clinical diagnosis of nephroblastoma rely mainly on clinical symptoms combined with ultrasound, IVU or CT. Although most cases can be diagnosed before operation, the prognosis is affected by missing opportunity to cure due to early detection failure . Therefore,to find a viable method for early diagnosis of nephroblastoma is very importan to its treatment and prognosis. With the development of proteomics, the desired serum protein markers as indicators for early screening and diagnosis are worthy of concern and research.Proteomics technology can high-throughput and rapidly screen protein profile expressed by gene in different phase of tumor so as to find a large number of protein biomarkers of diagnostic value, and these proteins could provide targets for tumor therapy, and provide effective tumor markers for early diagnosis. Surface-enhanced laser desorption / ionization-time of flight-mass spectrum ( SELDI-TOF-MS) protein chip technology has been widely used in the detection and screening of various types of disease-specific biomarkers, and made a series of breakthroughs. SELDI-TOF-MS technology as a new proteomics methods, with a small amount of samples, simple operation, high sensitivity, high-throughput, through a combination of MALDI-TOF-MS, LC-MS/MS, have identified tumor-specific serum proteins in breast cancer, ovarian cancer, pancreatic cancer and etc. In this study we find the specific protein markers by detecting serum samples of preoperative and postoperative patients and normal children using the SELDI-TOF MS technology, and they were further identified by mass Spectrometry techniques.ObjectiveTo screen and characterize the serum protein biomarkers in nephroblastoma, in order to establish the proteins as the specific serum biomarkers for diagnosis and prognosis monitoringMaterial and Methods1. Clinical Material A total of 130 serum samples were from pediatric surgery of the First Affiliated Hospital of Zhengzhou University: 30 preoperative nephroblastoma(stage I of 6 cases, stage II of 10 cases, stagelllof 10 cases, stage IV of 4 cases), 24 postoperative nephroblastoma (2 weeks after operation), 23 postoperative nephroblastoma (3 months and 6 months after operation respectively ). All of the nephroblastoma samples were confirmed by two pathologists. Patients consist of 16 boys and 14 girls. The range of age is from 24 days to 10 years old and the mean age is 2.8±0.1; 24 postoperative patients included 16 boys and 8 girls with the mean age of 2.2±0.1, and none has received radiotherapy or chemotherapy. The control group is composed of 30 healthy children from out-patient department. The age and sex of healthy individuals were matched with nephroblastoma group . All the blood species were drawn on empty stomach in the morning, and after the specimens are placed under 4℃for 1 hours, they are turned centrifugally for 10 minutes, and then the serum are extracted and preserved under -80℃.2. Main reagents and devicesCHAPS, urea, DTT, NaAC, SPA and trypsin were purchased from Promega Corporation (USA). Ciphergen PBS II + SELDI-TOF-MS and protein chip WCX2 were purchased from Ciphergen company(USA). Acetonitrile, CHCA, insulin, cytochrome C, IAM and NH4HCO3 were purchased from Sigma Corporation (USA).SHIMADZU LC-10Avp high-performance liquid chromatography (HPLC) were purchased from Shimadzu Corporation(Japan). Matrix-assisted laser desorption ionization/time of flight mass Spectrometry (AXIMA-CFRTM plus MALDI-TOF-MS) were purchased from Kratos Analytical Corporation (British). Centrifuge enrichment device, LC-MS/MS were purchased from Thermo electron Corporation (USA).3. Methods3.1 SELDI protein chip operations To unfreeze the serum specimens in ice bath, turn them centrifugally at 10000 rpm under 4℃for 2 minutes. Place a 96-hole plate on the ice box, add 10 ul of U9 (9MUrea, 2%CHAPS, 1%DTT) and 5 ul of serum to each hole, vibrate at 600 rpm under 4℃for 30 minutes in cold lab chamber. Before 15 minutes of finishing the vibration, pre-process the chip. The chip is placed in the Bioprocessor, note down the chip number, add 200 ul of NaNC (100 Mm, pH4) to each hole, vibrate at 600 rpm for 2 minutes in cold lab chamber, and repeat the above-mentioned operation once. Place the 96-hole plate being processed by U9 on the ice, use a medical gun to add 185 ul of NaNC, vibrate at 600 rpm under 4℃for 2 minutes in cold lab chamber. Add 100 ul of processed specimens to the chip, place them in the cold lab chamber under 4℃combining 600 rpm for 60 minutes, swing off the remaining liquid and dry out rapidly. Add 200 ul of NaAC, after vibrating at 600 rpm for 5 minutes, swing off and dry out, repeat this operation for three times. Wash each hole twice using 200 ul of deionized water, swing off the excessive water. After the chip is air dried, each hole is added 50% saturated SPA1 ul in two stages, after drying, place them on the device for testing.3.2 Purification of the differential protein peaks Take the serum samples out of the refrigerator(-80℃),then thawed in a water bath. After thawed , take 100μl serum, add 350μl ultrapure water, and then add 700μl pure acetonitrile. Put the above mixture in -20℃refrigerator, take it out 30 min later, centrifuge 10 min (13 000 r / min), and remove the supernatant into new tubes,then placed in SPD SpeedVacto freeze-dried about 20 min. Place the freeze-dried samples to HPLC. Collect the purified fluid at different times. Place the purified protein solution into the SPD SpeedVac to freeze-dried to about 20μl.Take CHCA and the above samples 1.5μl, and the mixture is spotted to the MALDI target plate . At the same time, use cytochrome C (relative molecular mass of 12361.96) + CHCA and insulin (molecular weight 5734.51) + CHCA as proof. Place the target plate into the MALDI-TOF-MS to detect the specific sample of the peak screened by SELDI-TOF-MS.3.3 Enzymolysis, mass Spectrometry analysis and database searchAdd ultrapure water to the specific protein samples to the volume of 40μl, adding with 4μl DTT (0.1 mol / L), and then placed in the warm water at 37℃for 1 h. Add 1.6μl IAM to the above 44μl solution and placed in the dark for 1 h. Add 1.6μl DTT (1 mol / L), 150μl NH4HCO3 (0.1 mol / L) and 2μl trypsm to the above 45.6μl solution, and then placed in the 37℃warm water overnight. Take the protein sample after digestion to the capillary column,and they were further analyzed by LC-MS/MS and the protein sequences were searched in database.4. Data collection and processing Use a protein chip whose molecular weight is known to adjust the SELDI-TOF-MS system, until the tolerance of molecular weight is less than 0.1%. Use mass spectrum reader to analyze the WCX2 protein chip combined with protein. Analysis parameters: laser strength is 170, sensitivity is 6, and the total number of each specimen collection is 140 times. The scope of data collection is 1000-30000 daltons; the optimized scope is 2000-20000 daltons. Use quality control serum to make repeated tests, the coefficients of variation of the peak value and the strength are 0.05% and 19.7% respectively.Use linear SVM classifier: The selection of feature vector uses the method of statistical filtration combining with model dependent screening, to build discrimination model, use the method of leave-one-out crossing verification to assess the discrimination result of the model.This experiment also uses discrimination analysis method to process mass spectrum data, to verify the result being processed by SVM.5. Statistical analysis After the original data of mass spectrum have been filtered out the noise and after clustering analysis, the mass-charge-ratio peaks data being preliminary screened out is carried out Wilcoxon rank sum test, the testing standard is set at a =0.01.Results1. SELDI-TOF-MS detection 11 M/Z peaks with a P value of less than 0.01 were obtained after processing mass spectral data of preoperative patients with Wilms' tumor and healthy controls. Subsequently, SVM was used to screen the combination model with the highest Youden index of the predicted value from any random combinations of proteins peaks with markedly different signal strength. Therefore, two protein markers with MZ ratios of 6984.4 and 6455.5were identified, whose expression levels were low in patients with Wilms' tumor ( the intensity: 1029.2±364.1,297.4±125.6) but were high in healthy children (the intensity: 2108.3±837.2, 753.4±225.8) . The two potential markers were together used in the cross validation by the leave-one out method and were found to have a specificity of 100% and a sensitivity of 100% for the discrimination model in the test set. Another peak with m/z of 9190.8 was weakly expressed in preoperative sera (the intensity: 283.4±153.9) but highly expressed in serum samples of postoperative patients (the intensity: 5973.6±656.7), and the difference does have statistics meaning (P< 0.01). The difference between postoperative group and normal children (the intensity: 5973.6±656.7, 6231.0±519.4) was not statistically significant(P> 0.01).2. Purification of differetial proteins After the serum of normal children (high expression of 6455.5 Da and 9190.8 Da) was separated and purified, a total of 25 different components of the protein solution were collected . By MALDI-TOF-MS detection, two proteins found in purified samples are the specific proteins of 6455.5Da and 9190.8 Da.3. Mass Spectrometry analysis and database search The protein at 6455.5Da and 9190.8Da were identified as apolipoprotein C-Ⅲand haptogloblin respectively. The matching rates of detected amino acid sequences of peptides in the database of people were 56.6% and 47.5% respectively. ConclusionThe detection of differentially expressed apolipoprotein C-Ⅲand haptogloblin may have utility for diagnosis,malignancy classification and prognosis monitoring of nephroblastoma and is worthy of further research and application.